The objectives of these studies are to analyze the molecular mechanisms underlying specific stage transitions in multistage carcinogenesis, comparing the mechanisms of promoting agents with those of carcinogens, singly or in combinations. This project addresses the primary question of how committed preneoplastic epithelial cells are irreversibly transformed by phosphorylation of specific proteins induced by the phospholipid- and calcium-dependent protein kinase (PK-C) triggered by pharmacologic diacylglycerol. Over 16 substrates for PK-C have been found in JB-6 cell lines. The reversibly associate with the particulate fraction of cells depending on the availability of divalent cations. Some are more readily phosphorylated under cation conditions favoring phosphorylation of more basic proteins (5.0 mM Ca++, 7.5 mM Mg++), but most are phosphorylated under high salt conditions (5.0 mM Ca++, 75 mM Mg++). One heat shock protein, pp80, is stimulated by TPA in untransformed but not transformed derivatives of JB-6 cells. Hyperthermia induces a full heat shock response and blocks TPA promotion. The relationship between this defect in heat shock protein regulation and the synergistic antitumor effects of interferon and hyperthermia is being pursued. The generation of reactive oxygen, expecially the superoxide anion, has been found to be a required event in TPA-induced promotion in JB-6 cells. Secondary free radicals implicated in this activity are OH- and lipid peroxides but hydrogen peroxide is only toxic.